Anna Petit

KIT expression in chromophobe renal cell carcinoma: Comparative immunohistochemical analysis of KIT expression in different renal cell neoplasms

The overexpression of c-Kit in chromophobe renal cell carcinoma (ChRCC) has been described by comparative gene expression analyses and has been proposed as a possible specific hallmark of this neoplasm. The aim of our study was to establish its immunohistochemical expression in a large series of ChRCC and to compare it with other renal neoplasms. In our study, immunohistochemical characterization of KIT was performed in 87 renal neoplasms including 25 cases of ChRCC, 13 cases of renal oncocytoma, and 39 renal cell carcinomas (21 cases of conventional RCC [CRCC], 8 cases of CRCC with granular cell differentiation, and 10 cases of papillary RCC [PRCC]). Eighty-eight percent ChRCC and 71% oncocytomas showed immunohistochemical expression of KIT, while the other types of RCC studied were all negative. The meaning of immunohistochemical expression of KIT in ChRCC and oncocytomas is still unknown, but its immunohistochemical staining appears to be useful in distinguishing ChRCC from PRCC, CRCC, and its granular cell variant. Moreover, our findings support current models that consider that there is a histopathogenic relationship between oncocytoma and ChRCC. Finally, it should be determined whether KIT plays a role in the tumorigenesis of ChRCC and oncocytoma and whether targeted therapy with STI-571, an inhibitor of KIT, could be effective in exceptional cases of ChRCC with metastatic extension or recurrence.

MSH6 and MUTYH Deficiency Is a Frequent Event in Early-Onset Colorectal Cancer

Purpose: Early-onset colorectal cancer (CRC) is suggestive of a hereditary predisposition. Lynch syndrome is the most frequent CRC hereditary cause. The MUTYH gene has also been related to hereditary CRC. A systematic characterization of these two diseases has not been reported previously in this population. Experimental Design: We studied a retrospectively collected series of 140 patients ≤50 years old diagnosed with nonpolyposis CRC. Demographic, clinical, and familial features were obtained. Mismatch repair (MMR) deficiency was determined by microsatellite instability (MSI) analysis, and immunostaining for MLH1, MSH2, MSH6, and PMS2 proteins. Germline MMR mutations were evaluated in all MMR-deficient cases. Tumor samples with loss of MLH1 or MSH2 protein expression were analyzed for somatic methylation. Germline MUTYH mutations were evaluated in all cases. BRAF V600E and KRAS somatic mutational status was also determined. Results: Fifteen tumors (11.4%) were MSI, and 20 (14.3%) showed loss of protein expression (7 for MLH1/PMS2, 2 for isolated MLH1, 3 for MSH2/MSH6, 7 for isolated MSH6, and 1 for MSH6/PMS2). We identified 11 (7.8%) germline MMR mutations, 4 in MLH1, 1 in MSH2, and 6 in MSH6. Methylation analysis revealed one case with somatic MLH1 methylation. Biallelic MUTYH mutations were detected in four (2.8%) cases. KRAS and BRAF V600E mutations were present in 39 (27.9%) and 5 (3.6%) cases, respectively. Conclusions: Loss of MSH6 expression is the predominant cause of MMR deficiency in early-onset CRC. Our findings prompt the inclusion of MSH6 and MUTYH screening as part of the genetic counseling of these patients and their relatives. Clin Cancer Res; 16(22); 5402–13. ©2010 AACR.

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5′ end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

Adhesion molecules α, β and γ‐catenin as prognostic factors of tumour progression in upper urinary tract urothelial tumours: the role of AKT‐P/GSK‐3β/β‐catenin pathway

To evaluate α, β and γ‐catenin expression in upper urinary tract urothelial tumours (UUTC) and determine their value as prognostic factors; to investigate the correlation between the catenin complex and the AKT pathway.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Expression and mutational analyses of KIT and PDGFR-α in sarcomatoid renal cell carcinoma

Sir: Sarcomatoid renal cell carcinoma (SRCC), first described by Farrow et al. in 1967, is currently not regarded as a distinct histological category but as a high-grade transformation occurring in different renal cell carcinoma (RCC) subtypes. Prognosis of patients with SRCC is very poor because of high stage at presentation and ineffective treatment in advanced stages. The report of KIT expression in sarcomatoid differentiation of RCC raises the potential use of Imatinib for the treatment of this neoplasm. This hypothesis is studied more deeply in this work. Imatinib is a well-known targeted therapy against tyrosine kinases BCR-ABL, KIT and platelet-derived growth factor receptor (PDGFR) that is effective in chronic myelogenous leukaemia (CML) and gastrointestinal stromal tumour (GIST). In GIST, KIT or PDGFR-a (PDGFRA) gain of function mutations play a crucial role in its pathogenesis and are responsible for the success of Imatinib. This example among others provides a model of how expression and oncogenic activation of tyrosine kinases sustains the rationale and clinical implementation of biological treatments. Taking this model as a reference, we assessed the immunohistochemical expression of KIT and PDGFRA, both targeted by Imatinib (KIT: A 4502; Dako, Carpinteria, CA, USA, dilution 1:100; and PDGFRA: sc-338; Santa Cruz Biotechnology, Santa Cruz, CA, USA, dilution 1:100) in a series of 16 SRCCs assessing the epithelial and the sarcomatoid part independently. The epithelial components of the SRCC cases were classified according to the World Health Organization classification 2002 into Clear cell RCC (nine cases), Papillary RCC (one case), Chromophobe RCC (five cases) and Unclassified RCC (one case). For KIT, the extent of immunoreactivity was semiquantitatively categorized as focal (<25% of tumour cells positive), moderate (25–50% of tumour cells positive) and diffuse (>50% of tumour cells positive). For PDGFRA, a score of immunoreactivity in three categories (negative, low and high expression) was made up taking into account extension and intensity (Table 1). The extent of positivity was also regarded semiquantitatively in the same manner as for KIT. The immunoreactivity of renal tubules served as a reference of intensity and lower intensities were regarded as weak and intensities higher or the same as renal tubules were considered strong. Higher expression of both tyrosine kinase receptors was observed in the sarcomatoid part (81.2% KIT and 81.2% PDGFRA) in comparison with the epithelial part (31.2% KIT and 43.7% PDGFRA) (Table 1, Figure 1). Of note, cases positive for KIT in the epithelial part were of Chromophobe RCC subtype in accordance with already published data (Figure 1 (A1)). Since the approval of Imatinib as an effective treatment against GIST and CML, many studies have been published characterizing expression of Imatinib targets on different neoplasms with the purpose of increasing the range of tumours in which Imatinib could be a therapeutic option. Recent data support the concept that mutation-mediated activation of these two tyrosine kinase receptors is a prerequisite for successful treatment with Imatinib and that sensitivity to Imatinib can not be inferred based solely on positive immunohistochemical expression of its targets. Based on these guidelines, we searched for mutational status of both KIT and PDGFRA in a subset SRCC cases where sarcomatoid differentiation showed overexpression of these proteins. In our analysis, we concentrated on exons 9 and 11 of KIT and 12 and 18 of PDGFRA, where activating mutations in KIT and PDGFRA have been most commonly found in GIST. Therefore, sequencing of KIT exons 9, 11 was done in the sarcomatoid part of six cases, two of which expressed KIT focally, one moderately and three diffusely. Mutational analysis of PDGFRA exons 12 and 18 was done in the sarcomatoid part of seven cases, all of which showed high expression of PDGFRA. None of the cases analysed harboured mutations in KIT or in PDGFRA in the sequenced exons. These results are in concordance with those of Sengupta et al., who reported absence of exon 9, 11, 13 and 17 KIT mutations in seven KIT positive high-grade RCCs, five of which exhibited sarcomatoid differentiation. In conclusion, our work shows that KIT and PDGFRA expression is a common finding in sarcomatoid differentiation of RCC. The molecular mechanism underlying this expression and whether this could have an active role in the clinical aggressiveness of this highgrade transformation is still unknown. We sought for specific mutations in KIT and PDGFR exons that have been involved in GIST tumorigenesis and response to

Alveolar soft part sarcoma presenting with cutaneous metastases: Report of a case with immunohistochemical and molecular characterization

Alveolar soft part sarcoma (ASPS) is an uncommon neoplasm of uncertain histogenesis that usually behaves as a painless, slow-growing mass that metastasizes early. We report a 21-year-old woman with cutaneous metastases of ASPS, whose histologic characteristics gave rise to a wide range of differential diagnoses of both primary and metastatic cutaneous neoplasms. The tumor failed to show a characteristic immunoprofile using routine immunohistochemical procedures, but was strongly and diffusely positive for the TFE3 antibody. The molecular study identified a type 2 alveolar soft part locus-transcription factor E3 (ASPL-TFE3) fusion, secondary to der(17)t(X;17)(p11.2;q25) translocation. A computed tomography scan performed after the diagnosis was made disclosed a 13-cm primary tumor in the left buttock. Cutaneous metastases presenting as the first sign of ASPS have not been reported previously. We emphasize the difficulties in making the diagnosis of ASPS when it presents in an unusual manner.

Role of HHV‐8 and mTOR pathway in post‐transplant Kaposi sarcoma staging

Kaposis sarcoma (KS) is one of the most frequent transplant related tumors. Several pathways are involved; however, the impact of the molecular phenotype associated to the tumor stage and the behavior‐depending resultant therapy is still unknown. The aim of our study was to analyze the role of HHV‐8 and mTOR pathway in tumor stages of skin KS after renal transplantation. Twelve renal transplant recipients with cutaneous KS from five transplant centers (1980–2007) under reduction of immunosuppression or conversion to mTOR inhibitor were included. The expression of HHV‐8, PTEN, TGFβ, VEGF, phospho‐mTOR, and phospho‐P70S6K in tumoral tissue was analyzed. KS lesions were classified as patch, plaque, and nodule state. HHV‐8 infection was found in all tissue samples. KS lesions showed high activation of VEGF, p‐mTOR and p‐P70S6K, low PTEN, and null TGFβ expression. The only pathway activated in a staging‐dependent manner was mTOR with higher p‐mTOR and p‐P70S6K expression in nodule versus patch stage. KS lesions disappeared after 5.24 months in all converted patients without any recurrence in 14.05 years of mean follow‐up. The activation of mTOR pathway according to KS stages supports the rational of the mTOR inhibitor in post‐transplant Kaposi.

Telomerase mRNA expression and immunohistochemical detection as a biomarker of malignant transformation in patients with inflammatory bowel disease

BACKGROUND Inflammatory bowel disease is a premalignant condition for developing colorectal cancer. Since a correlation has been suggested between telomere length, chromosomal instability and neoplastic transformation in this setting, we sought to investigate whether telomerase expression in colorectal mucosa may constitute a biomarker for malignant transformation in patients with inflammatory bowel disease. PATIENTS AND METHODS Forty-seven patients with inflammatory bowel disease with and without cancer or dysplasia were evaluated for human telomerase reverse transcriptase hTERT immunostaining in paraffin-embedded, formalin-fixed colorectal tissues. In addition, hTERT mRNA expression was assessed in fresh frozen specimens from a second set of 35 patients with inflammatory bowel disease at high or low risk for neoplastic transformation. RESULTS Five out of 10 patients (50%) with colorectal cancer or high-grade dysplasia exhibited hTERT immunochemical detection in adjacent, non-transformed colonic mucosa. However, this phenomenon was also observed in non-affected mucosa of patients with either long-standing (13 out of 19 patients; 68%) or short duration (13 out of 18 patients; 72%) disease without cancer or dysplasia. On the other hand, hTERT mRNA expression in non-affected colorectal mucosa from patients at high risk for neoplastic transformation due to long-standing disease was higher than in those at low risk (7.42+/-6.43 vs. 2.87+/-1.47, respectively; p=0.006). CONCLUSIONS Whereas hTERT immunostaining provides equivocal results, the observation that patients at high risk for colorectal cancer because of long-standing inflammatory bowel disease overexpress hTERT mRNA in non-affected colorectal mucosa suggests its potential usefulness as a biomarker of the risk of malignant transformation.